羧基改性多孔磁性壳聚糖微球共固定化葡萄糖氧化酶与过氧化氢酶

Glucose oxidase and catalase co-immobilized bycarboxyl modified porous magnetic chitosan microspheres

壳聚糖分子链上的活性基团数量有限,为了进一步增加壳聚糖的载酶量及固定化酶的生物催化稳定性,以3-氨丙基三乙氧基硅烷(APTES)和戊二酸酐(GA)为表面修饰剂,制备得到新型羧基功能化多孔磁性壳聚糖微球(carboxyl modified porous magnetic chitosan microspheres,PMCSM-COOH),成功共固定化了葡萄糖氧化酶(glucose oxidase,GOD)与过氧化氢酶(catalase,CAT)。研究表明,在PMCSM-COOH/GOD/CAT质量比为100/9.34/10.78时,GOD相对酶活达到最高为131.91%±0.58%。与游离酶相比,共固定化酶的温度稳定性、酸碱稳定性与储藏稳定性均有所提升,与底物的亲和力也有所增强,重复使用10次后仍保留着较高的相对酶活,为79.16%±1.98%。游离酶与固定化酶二级结构表明,固定化酶的α-螺旋相对含量高于游离GOD,这使得固定化酶具有更好的刚性结构,其稳定性优于游离酶。

The number of active groups on the molecular chain of chitosan is limited.In order to further increase the amount of chitosan enzyme and the biocatalysis stability of immobilized enzyme,in this study,3-aminopropyl triethoxysilane(APTES)and glutaric anhydride(GA)are used as surface modifiers to prepare carboxyl functionalized porous magnetic chitosan microspheres(PMCSM-COOH),and Glucose oxidase(GOD)and Catalase(CAT)are successfully immobilized.The results show that GOD relative enzyme activity reaches the highest value of(131.91±0.58)%when the mass ratio of PMCSM-COOH/GOD/CAT is 100/9.34/10.78.Compared with the free enzyme,the temperature stability,acid-base stability and storage stability of the co-immobilized enzyme are improved,and the affinity with the substrate is also enhanced.After repeated use for 10 times,the co-immobilized enzyme still retains a high relative enzyme activity of(79.16±1.98)%.The secondary structure of free enzyme and immobilized enzyme show that the relativeα-helix content of immobilized enzyme is higher than that of free GOD,so that immobilized enzyme has better structural rigidity and stability than free enzyme.